PROJECT SUMMARY In this application we address the need for new influenza drugs. Influenza, caused by influenza A and B viruses, is a major threat to human health and is associated with substantial healthcare costs every year. Although a vaccine is available, the vaccination rates remain low, and the vaccine is suboptimal in populations such as the elderly and immunocompromised who are most at risk of hospitalization and death due to influenza. As such, influenza drugs are an essential component of our influenza defense strategy, especially during a pandemic when vaccines are unavailable. However we have only one class of approved drug for influenza that is used clinically; the neuraminidase inhibitors, and there is growing concern that resistance may arise and render them ineffective. New influenza drugs are needed that have distinct targets and mechanisms from those of existing influenza drugs, and to this end we conducted a high throughput screen of almost 1 million molecules for the discovery of novel influenza virus inhibitors. This phenotypic screen resulted in 744 hits and the purpose of the current proposal is to perform hit validation on these compounds via medicinal chemistry triage and the use of multiple orthogonal biological assays. We aim to refine the hit list by eliminating scaffolds with reactive functionalities and obvious liabilities, and to validate the activity of re-purchased compounds in an orthogonal influenza antiviral assay. Through subsequent examination of multiple influenza A virus subtypes, oseltamivir-resistant virus, activity in primary human cells, and activity against influenza B virus, we will prioritize compounds with pan-influenza virus activity. Initial SAR studies will be performed with commercial analogs along with preliminary ADME studies, which will further identify any liabilities and facilitate selection of 3 leads for mechanism of action and target identification studies. Our overall goal is to identify 1-2 lead series of pan-influenza inhibitors with novel mechanisms that can serve as small molecule probes as well as leads for further optimization to drug candidates.